Treatment for stroke management

ABSTRACT

A method of treating a CNS injury to the brain, in a mammal in need of treatment, which comprises administering to said mammal an effective amount of a cytokine suppressive binding protein compound of formula I                    
     wherein the radicals are defined herein.

Applicant claims the benefit of Provisional application No. 60/103,320filed Oct. 7, 1998.

FIELD OF THE INVENTION

This invention relates to the novel use of imidazole compounds in thetreatment of stroke and stroke management.

BACKGROUND OF THE INVENTION

Interleukin-1 (IL-1) and Tumor Necrosis Factor (TNF) are biologicalsubstances produced by a variety of cells, such as monocytes ormacrophages. IL-1 has been demonstrated to mediate a variety ofbiological activities thought to be important in immunoregulation andother physiological conditions such as inflammation [See, e.g.,Dinarello et al., Rev. Infect. Disease, 6, 51 (1984)]. The myriad ofknown biological activities of IL-1 include the activation of T helpercells, induction of fever, stimulation of prostaglandin or collagenaseproduction, neutrophil chemotaxis, induction of acute phase proteins andthe suppression of plasma iron levels.

There are many disease states in which excessive or unregulated IL-1production is implicated in exacerbating and/or causing the disease.These include rheumatoid arthritis, osteoarthritis, endotoxemia and/ortoxic shock, syndrome, other acute or chronic inflammatory diseasestates such as the inflammatory reaction induced by endotoxin orinflammatory bowel disease; tuberculosis, atherosclerosis, muscledegeneration, cachexia, psoriatic arthritis, Reiter's syndrome,rheumatoid arthritis, gout, traumatic arthritis, rubella arthritis, andacute synovitis. Recent evidence also links IL-1 activity to diabetesand pancreatic β cells.

Dinarello, J. Clinical Immunology, 5 (5), 287-297 (1985), reviews thebiological activities which have been attributed to IL-1. It should benoted that some of these effects have been described by others asindirect effects of IL-1.

Excessive or unregulated TNF production has been implicated in mediatingor exacerbating a number of diseases including rheumatoid arthritis,rheumatoid spondylitis, osteoarthritis, gouty arthritis and otherarthritic conditions; sepsis, septic shock, endotoxic shock, gramnegative sepsis, toxic shock syndrome, adult respiratory distresssyndrome, cerebral malaria, chronic pulmonary inflammatory disease,silicosis, pulmonary sarcoisosis, bone resorption diseases, reperfusioninjury, graft vs. host reaction, allograft rejections, fever andmyalgias due to infection, such as influenza, cachexia secondary toinfection or malignancy, cachexia, secondary to acquired immunedeficiency syndrome (AIDS), AIDS, ARC (AIDS related complex), keloidformation, scar tissue formation, Crohn's disease, ulcerative colitis,or pyresis.

Interleukin-8 (IL-8) is a chemotactic factor first identified andcharacterized in 1987. IL-8 is produced by several cell types includingmononuclear cells, fibroblasts, endothelial cells, and keratinocytes.Its production from endothelial cells is induced by IL-1, TNF, orlipopolysachharide (LPS). Human IL-8 has been shown to act on Mouse,Guinea Pig, Rat, and Rabbit Neutrophils. Many different names have beenapplied to IL-8, such as neutrophil attractant/activationprotein-1(NAP-1), monocyte derived neutrophil chemotactic factor(MDNCF), neutrophil activating factor (NAF), and T-cell lymphocytechemotactic factor.

IL-8 stimulates a number of functions in vitro. It has been shown tohave chemoattractant properties for neutrophils, T-lymphocytes, andbasophils. In addition it induces histamine release from basophils fromboth normal and atopic individuals as well as lysozomal enzyme releaseand respiratory burst from neutrophils. IL-8 has also been shown toincrease the surface expression of Mac-1 (CD11b/CD18) on neutrophilswithout de novo protein synthesis, this may contribute to increasedadhesion of the neutrophils to vascular endothelial cells. Many diseasesare characterized by massive neutrophil infiltration.

IL-1 and TNF affect a wide variety of cells and tissues and thesecytokines as well as other leukocyte derived cytokines are important andcritical inflammatory mediators of a wide variety of disease states andconditions. The inhibition of these cytokines is of benefit incontrolling, reducing and alleviating many of these disease states.

There remains a need for the treatment, and for the prevention of CNSinjuries which are related to the ability of compounds which arecytokine suppressive, i.e. compounds which are capable of inhibitingcytokines, such as IL-1, IL-6, IL-8 and TNF.

SUMMARY OF THE INVENTION

This invention relates to the use of substituted imidazole compounds, orpharmaceutical compositions thereof in the treatment of stroke, andstroke management.

The compounds for use herein are described in PCT[US97/09888 filed Jun.6, 1997, and published Dec. 18, 1997 as WO 97/47618, Liverton et al.,now U.S. Pat. No. 5,859,041 granted Jan. 12, 1999, whose disclosures areincorporated herein by reference in their entirety.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is to the novel use of the compounds described inWO 97/47618 for the treatment, an acute setting, as well as includingprophylactic use, in preventing in those individuals deemed susceptibleto, various CNS injuries.

Synthetic chemistry, dosages, and methods of making pharmaceuticalformulations thereof are also contained within the noted patentapplication.

Another aspect of the present invention relates to the novel use ofcompounds described in WO 98/47899 (PCT/US98/07831), Dodd et al., forthe treatment, an acute setting, as well as including prophylactic use,in preventing in those individuals deemed susceptible to, various CNSinjuries. Synthetic chemistry, dosages, and methods of makingpharmaceutical formulations thereof are also contained within the notedpatent application.

Another aspect of the present invention relates to the novel use ofcompounds described in WO 98/52937 (PCT/US98/10807), Anantanarayan etal., now U.S. Pat. No. 5,932,576, granted Aug. 3, 1999; U.S. Pat. No.6,0874,96, and U.S. Pat. No. 6,335,336 for the treatment, an acutesetting, as well as including prophylactic use, in preventing in thoseindividuals deemed susceptible to, various CNS injuries. Syntheticchemistry, dosages, and methods of making pharmaceutical formulationsthereof are also contained within the noted patent and patentapplication.

Another aspect of the present invention relates to the novel use ofcompounds described in WO 99/03837 (PCT/US98/13419), Wachter et al., nowU.S. Pat. No. 6,040,320 for the treatment, an acute setting, as well asincluding prophylactic use, in preventing in those individuals deemedsusceptible to, various CNS injuries. Synthetic chemistry, dosages, andmethods of making pharmaceutical formulations thereof are also containedwithin the noted patent application.

Another aspect of the present invention relates to the novel use ofcompounds described in WO 98/47892 (PCT/US98/07910), Beers et al., nowU.S. Pat. No. 5,965,583 and U.S. Pat. No. 6,214,830 for the treatment,an acute setting, as well as including prophylactic use, in preventingin those individuals deemed susceptible to, various CNS injuries.Synthetic chemistry, dosages, and methods of making pharmaceuticalformulations thereof are also contained within the noted patentapplication.

Another aspect of the present invention relates to the novel use ofcompounds described in WO 99/01449 (PCT/EP98/03930), Revesz et al., nowU.S. Pat. No. 6,300,347 for the treatment, an acute setting, as well asincluding prophylactic use, in preventing in those individuals deemedsusceptible to, various CNS injuries. Synthetic chemistry, dosages, andmethods of making pharmaceutical formulations thereof are also containedwithin the noted patent application.

Another aspect of the present invention relates to the novel use ofcompounds described in WO 98/52941 (PCT/US98/11684), Hanson et al., nowU.S. Pat. No. 6,087,381 for the treatment, an acute setting, as well asincluding prophylactic use, in preventing in those individuals deemedsusceptible to, various CNS injuries. Synthetic chemistry, dosages, andmethods of making pharmaceutical formulations thereof are also containedwithin the noted patent application.

Another aspect of the present invention relates to the novel use ofcompounds described in WO 98/52941 (PCT/US98/11684), Hanson.et al., forthe treatment, an acute setting, as well as including prophylactic use,in preventing in those individuals deemed susceptible to, various CNSinjuries. Synthetic chemistry, dosages, and methods of makingpharmaceutical formulations thereof are also contained within the notedpatent application.

Another aspect of the present invention relates to the novel use ofcompounds described in WO 97/05878 (PCT/US96/12922), De Laszlo et al;,now U.S. Pat. No. 5,837,719, granted Nov. 17, 1998, for the treatment,an acute setting, as well as including prophylactic use, in preventingin those individuals deemed susceptible to, various CNS injuries.Synthetic chemistry, dosages, and methods of making pharmaceuticalformulations thereof are also contained within the noted patent andpatent application.

Another aspect of the present invention relates to the novel use ofcompounds described in WO 97/16441 (PCT/US96/17324), De Laszlo et al.,for the treatment, an acute setting, as well as including prophylacticuse, in preventing in those individuals deemed susceptible to, variousCNS injuries. Synthetic chemistry, dosages, and methods of makingpharmaceutical formulations thereof are also contained within the notedpatent application.

Another aspect of the present invention relates to the novel use ofcompounds described in WO 98/21957 (PCT/US97/21019), Chang et al., nowU.S. Pat. No. 5,955,480 for the treatment, an acute setting, as well asincluding prophylactic use, in preventing in those individuals deemedsusceptible to, various CNS injuries. Synthetic chemistry, dosages, andmethods of making pharmaceutical formulations thereof are also containedwithin the noted patent application.

Another aspect of the present invention relates to the novel use ofcompounds described in WO 97/12876 (PCT/US96/15880), now U.S. Pat. No.5,717,100, granted Feb. 10, 1998, Selnick et al., and U.S. Pat. No.6,083,949 for the treatment, an acute setting, as well as includingprophylactic use, in preventing in those individuals deemed susceptibleto, various CNS injuries. Synthetic chemistry, dosages, and methods ofmaking pharmaceutical formulations thereof are also contained within thenoted patent application.

Another aspect of the present invention relates to the novel use ofcompounds described in WO 97/16426 (PCT/US96/17477), De Laszlo et al.,for the treatment, an acute setting, as well as including prophylacticuse, in preventing in those individuals deemed susceptible to, variousCNS injuries. Synthetic chemistry, dosages, and methods of makingpharmaceutical formulations thereof are also contained within the notedpatent application.

Another aspect of the present invention relates to the novel use ofcompounds described in WO 97/05877 (PCT/US96/12917), De Laszlo et al.,now U.S. Pat. No. 5,782,778, granted Aug. 11, 1998, for the treatment,an acute setting, as well as including prophylactic use, in preventingin those individuals deemed susceptible to, various CNS injuries.Synthetic chemistry, dosages, and methods of making pharmaceuticalformulations thereof are also contained within the noted patentapplication.

Another aspect of the present invention relates to the novel use ofcompounds described in WO 97/16442 (PCT/US96/18539), De Laszlo et al.,for the treatment, an acute setting, as well as including prophylacticuse, in preventing in those individuals deemed susceptible to, variousCNS injuries. Synthetic chemistry, dosages, and methods of makingpharmaceutical formulations thereof are also contained within the notedpatent application.

Another aspect of the present invention relates to the novel use ofcompounds described in WO 98/52940 (PCT/US98/10436), Anantanarayan etal., now U.S. Pat. No. 6,423,713 for the treatment, an acute setting, aswell as including prophylactic use, in preventing in those individualsdeemed susceptible to, various CNS injuries. Synthetic chemistry,dosages, and methods of making pharmaceutical formulations thereof arealso contained within the noted patent application.

Another aspect of the present invention relates to the novel use ofcompounds described in WO 99/20624 (PCT/EP98/06472) Cheng et al., nowU.S. Pat. No. 6,316,464, U.S. Pat. No. 6,479,507 for the treatment, anacute setting, as well as including prophylactic use, in preventing inthose individuals deemed susceptible to, various CNS injuries. Syntheticchemistry, dosages, and methods of making pharmaceutical formulationsthereof are also contained within the noted patent application.

CNS injuries as defined herein include both open or penetrating headtrauma, such as by surgery, or a closed head trauma injury, such as byan injury to the head region. Also included within this definition isischemic stroke, particularly to the brain area.

Ischemic stroke may be defined as a focal neurologic disorder thatresults from insufficient blood supply to a particular brain area,usually as a consequence of an embolus, thrombi, or local atheromatousclosure of the blood vessel. The role of inflammatory cytokines in thisare has been emerging and the present invention provides a mean for thepotential treatment of these injuries. Relatively little treatment, foran acute injury such as these has been available.

In addition to the advantage of using a compound which is an inhibitorof cytokine suppressive binding protein activity (CSBP) for theinhibition of IL-1 or TNF alone which is neuroprotective, inhibition ofboth cytokines provides for increased efficacy. These compounds may alsobe useful for all types of strokes, regardless of thethrombotic/hemorrhagic variants. This would allow for earlyintervention, possible without use of CAT scans, and potential use inconjugation with tPA, or streptokinase, for instance.

TNF-α is a cytokine with proinflammatory actions, including endothelialleukocyte adhesion molecule expression. Leukocytes infiltrate intoischemic brain lesions and hence compounds which inhibit or decreaselevels of TNF would be useful for treatment of ischemic brain injury.See Liu et al., Stoke, Vol. 25.; No. 7, pp. 1481-88 (1994) whosedisclosure is incorporated herein by reference.

Models of closed head injuries and treatment with mixed 5-LO/CO agentsis discussed in Shohami et al., J. of Vaisc & Clinical Physiology andPharmacology, Vol. 3, No. 2, pp. 99-107 (1992) whose disclosure isincorporated herein by reference. Treatment which reduced edemaformation was found to improve functional outcome in those animalstreated.

As noted, these compounds are useful as cytokine inhibitors, and inparticular the preferred method of inhibition is the inhibition of theCSBP/p38/RK kinase pathway. A description of the assay for inhibition ofthe cytokine specific binding protein (CSBP) is also found in WO95/07922, whose disclosure is incorporated by reference in its entirety,as well as in U.S. Pat. No. 5,777,097. A kinase binding assay is alsodescribed herein. Updated versions of a CSBP kinase assay may be foundin later filed SB patent applications including WO 98/57966 forinstance.

The compounds of WO 97/47618 can be used in the manufacture of amedicament for the prophylactic or therapeutic treatment of any diseasestate in a human, or other mammal, which is exacerbated or caused by aneurotraumatic event, such as closed head injuries.

In addition, the compounds of WO 99/20624, WO 98/52940, WO 97/16442, WO97/05877, WO 97/16426, WO 97/12876, WO 98/21957, WO 97/16441, WO97/05878, WO 98/52941, WO 99/01449, WO 98/47892, WO 99/03837, WO98/52937, and WO 98/47899, can be used in the manufacture of amedicament for the prophylactic or therapeutic treatment of any diseasestate in a human, or other mammal, which is exacerbated or caused by aneurotraumatic event, such as closed head injuries.

The compounds of these patent applications are capable of inhibitingproinflammatory cytokines, such as IL-1, IL-6, IL-8 and TNF and aretherefore of use in therapy. IL-1, IL-6, IL-8 and TNF affect a widevariety of cells and tissues and these cytokines, as well as otherleukocyte-derived cytokines, are important and critical inflammatorymediators of a wide variety of disease states and conditions. Theinhibition of these pro-inflammatory cytokines is of benefit incontrolling, reducing and alleviating many of these disease states.

Accordingly, the present invention provides for a method of treating aneurotraumatic disease, in a mammal in need thereof, which methodcomprises administering to said mammal an effective amount of a CSAID™,cytokine suppressive inhibitory compound, wherein the compound is aninhibitor of the CSBP/p38/RK kinase.

As used herein, the term “inhibiting the production of IL-1 (IL-6, IL-8or TNF)” refers to:

a) a decrease of excessive in 3in vivo levels of the cytokine (IL-1,IL-6, IL-8 or TNF) in a human to normal or sub-normal levels byinhibition of the in vivo release of the cytokine by all cells,including but not limited to monocytes or macrophages;

b) a down regulation, at the genomic level, of excessive in vivo levelsof the cytokine (IL-1, IL-6, IL-8 or TNF) in a human to normal orsub-normal levels;

c) a down regulation, by inhibition of the direct synthesis of thecytokine (IL-1, IL-6, IL-8 or TNF) as a postranslational event; or

d) a down regulation, at the translational level, of excessive in vivolevels of the cytokine (IL-1, IL-6, IL-8 or TNF) in a human to normal orsub-normal levels.

As used herein, the term “cytokine interfering” or “cytokine suppressiveamount” refers to an effective amount of a compound of Formula (I) whichwill cause a decrease in the in vivo levels of the cytokine to normal orsub-normal levels, when given to a patient for the prophylaxis ortreatment of a disease state which is exacerbated by, or caused by,excessive or unregulated cytokine production.

A new member of the MAP kinase family, alternatively termed CSBP, p38,or RK, has been identified, See Lee et al., Nature, Vol. 300 n(72),739-746 (1994). Activation of this novel protein kinase via dualphosphorylation has been observed in different cell systems uponstimulation by a wide spectrum of stimuli, such as physicochemicalstress and treatment with lipopolysaccharide or proinflammatorycytokines such as interleukin-1 and tumor necrosis factor. The cytokinebiosynthesis inhibitors, of the present invention, compounds of Formula(I), have been determined to be potent and selective inhibitors ofCSBP/p38/RK kinase activity. These inhibitors are of aid in determiningthe signaling pathways involvement in inflammatory responses.

The invention will now be described by reference to the followingbiological examples which are merely illustrative and are not to beconstrued as a limitation of the scope of the present invention.

In vivo TNF Assay:

(1) Griswold et al., Drugs Under Exp. and Clinical Res., XIX (6),243-248 (1993); or

(2) Boehm, et al., Journal Of Medicinal Chemistry 39, 3929-3937 (1996)whose disclosures are incorporated by reference herein in theirentirety.

LPS-induced TNFα Production in Mice and Rats

In order to evaluate in vivo inhibition of LPS-induced TNFα productionin rodents, both mice and rats are injected with LPS.

Mouse Method,

Male Balb/c mice from Charles River Laboratories are pretreated (30minutes) with compound or vehicle. After the 30 min. pretreat time, themice are given LPS (lipopolysaccharide from Esherichia coli Serotype055-85, Sigma Chemical Co., St Louis, Mo.) 25 ug/mouse in 25 ulphosphate buffered saline (pH 7.0) intraperitoneally. Two hours laterthe mice are killed by CO₂ inhalation and blood samples are collected byexsanguination into heparinized blood collection tubes and stored onice. The blood samples are centrifuged and the plasma collected andstored at −20° C. until assayed for TNFα by ELISA.

Rat Method

Male Lewis rats from Charles River Laboratories are pretreated atvarious times with compound or vehicle. After a determined pretreattime, the rats are given LPS (lipopolysaccharide from Esherichia coliSerotype 055-85, Sigma Chemical Co., St Louis, Mo.) 3.0 mg/kgintraperitoneally. The rats are killed by CO₂ inhalation and heparinizedwhole blood is collected from each rat by cardiac puncture 90 minutesafter the LPS injection. The blood samples are centrifuged and theplasma collected for analysis by ELISA for TNFα levels.

ELISA Method

TNFα levels are measured using a sandwich ELISA, as described in Oliveraet al., Circ. Shock, 37, 301-306, (1992), whose disclosure isincorporated by reference in its entirety herein, using a hamstermonoclonal antimurine TNFα (Genzyme, Boston, Mass.) as the captureantibody and a polygonal rabbit antimurine TNFa (Genzyme) as the secondantibody. For detection, a peroxidase-conjugated goat antirabbitantibody (Pierce, Rockford, Ill.) is added, followed by a substrate forperoxidase (1 mg/ml orthophenylenediamine with 1% urea peroxide). TNFαlevels in the plasma samples from each animal are calculated from astandard curve generated with recombinant murine TNFα (Genzyme).

LPS-Stimulated Cytokine Production in Human Whole Blood

Assay: Test compound concentrations are, prepared at 10×concentrationsand LPS prepared at 1 ug/mI (final conc. of 50 ng/ml LPS) and added in50 uL volumes to 1.5 mL eppendorf tubes. Heparinized human whole bloodis obtained from healthy volunteers and is dispensed into eppendorftubes containing compounds and LPS in 0.4 mL volumes and the tubesincubated at 37 C. Following a 4 hour incubation, the tubes arecentrifuged at 5000 rpm for 5 minutes in a TOMY microfuge, plasma iswithdrawn and frozen at −80 C.

Cytokine measurement: IL-1and/or TNF were quantified using astandardized ELISA technology. An in-house ELISA kit was used to detecthuman IL-1 and TNF. Concentrations of IL-1 or TNF were determined fromstandard curves of the appropriate cytokine and IC50 values for testcompound (concentration that inhibited 50% of LPS-stimulated cytokineproduction) were calculated by linear regression analysis.

CSBP/p38 Kinase Assay:

This assay measures the CSBP/p38-catalyzed transfer of ³²P from[a-³²P]ATP to threonine residue in an epidermal growth factor receptor(EGFR)-derived peptide (T669) with the following sequence:KRELVEPLTPSGEAPNQALLR (residues 661-681). (See Gallagher et al.,“Regulation of Stress Induced Cytokine Production by PyridinylImidazoles: Inhibition of CSBP Kinase”, BioOrganic & MedicinalChemistry, 1997, 5, 49-64).

Reactions are carried in round bottom 96 well plate (from Corning) in a30 ml volume. Reactions contained (in final concentration): 25 mM Hepes,pH 7.5; 8 mM MgCl₂; 0.17 mM ATP (the Km[_(ATP)] of p38 (see Lee et al.,Nature 300, n72 pg. 639-746 (December 1994)); 2.5 uCi of [g-32P]ATP; 0.2mM sodium orthovanadate; 1 mM DTT; 0.1% BSA; 10% glycerol; 0.67 mM T669peptide; and 2-4 nM of yeast-expressed, activated and purified p38.Reactions are initiated by the addition of [gamma-32P]Mg/ATP, andincubated for 25 min. at 37° C. Inhibitors (dissolved in DMSO) areincubated with the reaction mixture on ice for 30 minutes prior toadding the 32P-ATP. Final DMSO concentration was 0. 16%. Reactions areterminated by adding 10 ul of 0.3 M phosphoric acid, and phosphorylatedpeptide is isolated from the reactions by capturing it on p81phosphocellulose filters. Filters are washed with 75 mM phosphoricacids, and incorporated 32P was quantified using beta scintillationcounter. Under these conditions, the specific activity of p38 is about400-450 pmol/pmol enzyme, and the activity linear for up to 2 hr ofincubation. The kinase activity values are obtained after subtractingvalues generated in the absence of substrate which are 10-15% of totalvalues.

Prostoglandin Endoperoxide Synthase-2 (PGHS-2)assay:

This assay describes a method for determining the inhibitory effects ofcompounds of Formula (1) on human PGHS-2 protein expression in LPSstimulated human monocytes. A suitable assay for PGHS-2 proteinexpression may be found in a number of publications, including U.S. Pat.No. 5,593,992 whose disclosure is incorporated herein by reference.

TNF-a in Traumatic Brain Injury Assay

This assay provides for examination of the expression of tumor necrosisfactor mRNA in specific brain regions which follow experimentallyinduced lateral fluid-percussion traumatic brain injury (TBI) in rats.Since TNF-α is able to induce nerve growth factor (NGF) and stimulatethe release of other cytokines from activated astrocytes, thispost-traumatic alteration in gene expression of TNF-α plays an importantrole in both the acute and regenerative response to CNS trauma. Asuitable assay may be found in WO 97/35856 whose disclosure isincorporated herein by reference.

CNS Injury Model for IL-b mRNA

This assay characterizes the regional expression of interleukin- 1β(IL-1β) mRNA in specific brain regions following experimental lateralfluid-percussion traumatic brain injury (TBI) in rats. Results fromthese assays indicate that following TBI, the temporal expression ofIL-1β mRNA is regionally stimulated in specific brain regions. Theseregional changes in cytokines, such as IL-1β play a role in thepost-traumatic pathologic or regenerative sequelae of brain injury. Asuitable assay may be found in WO 97/35856 whose disclosure isincorporated herein by reference.

Evaluation of compounds in specific models for rat focal Ischemic Strokeinjury, may be determined by techniques well known to the skilledartisan, as described in some of the publications below. Alternatively,using microsurgical electrocoagulation of the middle cerebral arteryunder stereotaxic control, a suitable candidate compound is administeredby either intravenous, or oral administration. Pose 24 hours of MCAO,the neurological deficits and stained forebrain sections are evaluated.Suitable endpoints are 1) neurological deficits in the forelimb, andhindlimb; 2) infact volume of (mm3); and hemispheric infarct (%;normalized to normal contralateral hemisphere).

Additional publication which are of value in this invention include,Barone et al., Stroke, 28:6, 1233-1244 (1997); Stroke, 23:9, 1337-1347(1997); Stroke, 25:7, 1481-1488 (1994); Stroke, 24:11, 1746-51 (1993);Stroke, 28:1, 155-162 (1997); Stroke 26:9, 1665-1669 (1995);Cerebrovascular & Brain Metabolism Reviews, 6:4, 341-360 (1994); BrainResearch Bulletin, 35:4, 387-392 (1994); Brain Research Bulletin, 31:565-572 (1993); J. Cerberal Blood Flow and Metabolism, 16:3, 260-366(1996); Neuroscience and Biobehavioral Reviews, 20:3, 445-452 (1996);Cerebrovascular Diseases: 19th Princeton Stroke Conference, Ed.Moskowitz et al., Boston: Butterworth-Heinemann Press, Chapter 7, pp.75-91 (1995); Immunotherapy in Neuroimmunologic Diseases, Editors, Zhanget al., Martin Dunitz, Ltd. 1998 (pp. 155-174); Molecular and ChemicalNeuropathy, 24: 13-30 (1995); Annals of N.Y. Academy of Sciences, Vol.825 (1997), Neuroprotective Agents, 3rd International Conference,Editors, Slikker et al., p. 179-193.

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference herein as thoughfully set forth.

The above description fully discloses the invention including preferredembodiments thereof. Modifications and improvements of the embodimentsspecifically disclosed herein are within the scope of the followingclaims. Without further elaboration, it is believed that one skilled inthe are can, using the preceding description, utilize the presentinvention to its fullest extent. Therefore, the Examples herein are tobe construed as merely illustrative and not a limitation of the scope ofthe present invention in any way. The embodiments of the invention inwhich an exclusive property or privilege is claimed are defined asfollows.

What is claimed is:
 1. A method of treating a CNS injury to the brain,in a mammal in need of such treatment, which method comprisesadministering to said mammal an effective amount of a cytokinesuppressive binding protein compound represented by the formula:

or a pharmaceutically acceptable salt thereof, wherein: X and X′ eachindependently represent —(CH₂)_(m) —Y—(CH₂)_(n—), wherein m and nrepresent integers within the range of from 0-4, such that the sum of mand n is from 0-6; Y represents a member selected from the groupconsisting of: a direct bond; O; S(O)_(y), with y equal to 0, 1 or 2;NR^(q)′, with R^(q)′ as defined below; C(O); OC(O); C(O)O; SO_(x)NR^(q)′with x equal to 1 or 2 and R^(q)′ as defined below; NR^(q)′SO_(x);C(O)NR^(q)′ and NR^(q)′ C(O);

represents a 4 to 10 membered non-aromatic heterocycle containing atleast one N atom, and optionally containing 1-2 additional N atoms and0-1 O or S atom; R^(x) represents H, C₁₋₆alkyl(R^(q))₃, C₃₋₈cycloalkyl,OC₁₋₆alkyl(R^(q))₃ or C(O)C₁₋₆alkyl(R^(q))₃; each R independentlyrepresents a member selected from the group consisting of: halo;hydroxy; C₁₋₆alkyl(R^(q))₃; OC₁₋₆alkyl(R^(q))₃; C₃₋₈cycloalkyl(R^(q))₃;CN; CONH₂; CONHC₁₋₆alkyl(R^(q))₃; CON(C₁₋₆alkyl(R^(q))₃)₂; NH₂;NHC₁₋₆alkyl(R^(q))₃; NHC₃₋₈cycloalkyl; N(C₁₋₆alkyl(R^(q))₃)₂;CON(C₃₋₈cycloalkyl)(C₁₋₆alkyl(R^(q))₃); CO)₂H; CO₂C₁₋₆alkyl(R^(q))₃;C(O)C₁₋₆alkyl(R^(q))₃; aryl(R^(q))₃; heterocyclyl(R^(q))₃;heteroaryl(R^(q))₃; CF₃; SH; NO₂; NHSO₂C₁₋₆alkyl(R^(q))₃; NHSO₂aryl(R^(q))₃, NHSO₂ heteroaryl(R^(q))₃; N(R^(q)′)C(O) C₁₋₆alkyl(R^(q))₃;NR^(q)′C(O)NH(C₁₋₆alkyl(R^(q))₃); C₂₋₄alkenyl(R^(q))₂₋₃ andC₂₋₄alkynyl(R^(q))₁₋₃; each R″ independently represents a memberselected from the group consisting of: halo; hydroxy; CC₁₋₆alkyl(R^(q))₃; OC₁₋₆alkyl(R^(q))₃; C₃₋₈cycloalkyl(R^(q))₃; CN;CONH₂; CONHC₁₋₆alkyl(R^(q))₃; CON(C₁₋₆alkyl(R^(q))₃)₂; NH₂;NHC₁₋₆alkyl(R^(q))₃; NHC₃₋₈cycloalkyl; N(C₁₋₆alkyl(R^(q))₃)₂;CON(C₃₋₈cycloalkyl)(C₁₋₆alkyl(R^(q))₃); CO₂H; CO₂C₁₋₆alkyl(R^(q))₃;C(O)C₁₋₆alkyl(R^(q))₃; aryl(R^(q))₃; heterocyclyl(R^(q))₃;heteroaryl(R^(q))₃; CF₃; SH; NO₂; SO_(y) C₁₋₆alkyl(R^(q))₃, with y asdefined above; SO₂NH₂; SO₂NHC₁₋₆alkyl(R^(q))₃; SO₂N(C₁₋₆alkyl(R^(q))₃)₂;NHSO₂C₁₋₆alkyl(R^(q))₃; NHSO₂aryl(R^(q))₃, NHSO₂ heteroaryl(R^(q))₃;N(R^(q))C(O)C₁₋₆alkyl(R^(q))₃; NR^(q)′C(O)NH(C₁₋₆alkyl(R^(q))₃);C₂₋₄alkenyl(R^(q))₂₋₃ and C₂₋₄alkynyl(R^(q))₁₋₃; each R′ independentlyrepresents a member selected from the group consisting of: CONH₂;CONHC₁₋₆alkyl(R^(q))₃3; CON(C₁₋₆alkyl(R^(q))₃)₂;CONHC₃₋₈cycloalkyl(R^(q))₃; CON(C₃₋₈cycloalkyl(R^(q))₃)₂;CON(C₃₋₈cycloalkyl)(C₁₋₆alkyl(R^(q))₃)); CO₂H; CO₂C₁₋₆alkyl(R^(q))₃;C(O)C₁₋₆alkyl(R^(q))₃; CO₂C₃₋₈cycloalkyl(R^(q))₃;C(O)C₃₋₈cycloalkyl(R^(q))₃; —[C(O)(CH₂)_(j)—CR⁵R⁶—(CH₂)_(k)—NR⁷]_(p)—R⁸;—C(O)heterocyclyl(R^(q))₃,—CON[C₁₋₆alkyl(R^(q))₃]C₃₋₈cycloalkyl(R^(q))₃]; —C(O)aryl(R^(q))₃;—C(O)heteroaryl(R^(q))₃; hydroxy; C₁₋₆alkyl(R^(q))₃;C₃₋₈cycloalkyl(R^(q))₃; OC₁₋₆alkyl(R^(q))₃; OC₃₋₈cycloalkyl(R^(q))₃;heterocyclyl(R^(q))₃; CN; NH(R^(q)″); NHC₁₋₆alkyl(R^(q))₃;N(C₁₋₆alkyl(R^(q))₃)₂; NHC₃₋₈cycloalkyl(R^(q))₃;N(C₃₋₈cycloalkyl(R^(q))₃)₂; CF₃; SH; NO₂; C₂₋₄alkenyl(R^(q))₂₋₃,aryl(R^(q))₃)₂; heteroaryl(R^(q))₃; C₂₋₄alkynyl(R^(q))₁₋₃;OC(O)C₃₋₈cycloalkyl(R^(q))₃; SO₂NH₂; SO₂NH C₁₋₆alkyl(R^(q))₃;SO₂N(C₁₋₆alkyl(R^(q))₃)₂; NH SO₂C₁₋₆alkyl(R^(q))₃; NHSO₂aryl(R^(q))₃;NHSO₂heteroaryl(R^(q))₃; OC(O)heterocyclyl(R^(q))₃; N(R^(q)′)C(O)C₁₋₆alkyl(R^(q))₃; NR^(q)′C(O)NH(C₁₋₆alkyl(R^(q))₃;OC(O)C₁₋₆alkyl(R^(q))₃; —OC(O)aryl(R^(q))₃; OC(O)heteroaryl(R^(q))₃;—C(═NR^(q)′)NH₂; —C(═NR^(q)′)NH C₁₋₆alkyl(R^(q))₃;—C(═NR^(q)′)N(C₁₋₆alkyl(R^(q))₃)₂;—O—[C(O)—(CH₂)_(j)—CR⁵R⁶—(CH₂)_(k)—NR⁷]_(p)-R⁸ and —[NR⁷(CH₂)_(k)—CR⁵R⁶—(CH₂)_(j) —C(O)]_(p)—OR⁹ wherein j and k independently representintegers of from 0-3; R⁵ and R⁶ are independently H, aryl,C₁₋₆alkyl(R^(q))₃, or CR⁵R⁶ in combination represents a 3, 4, 5 or 6membered cycloalkyl or heterocyclyl group, an aryl group or a heteroarylgroup; p represents 1, 2 or 3, with the proviso that when p represents1, CR⁵R⁶ represents a 3, 4, 5 or 6 membered cycloalkyl group or aheterocyclyl group, an aryl group or a heteroaryl group, and at leastone of j and k is 1, 2 or 3; R⁷ and R⁸ are independently H, C₁₋₆ alkylor aryl; R⁹ represents H, a negative charge balanced by a positivelycharged group or a protecting group; R^(q)represents a member selectedfrom the group consisting of: R^(q)′; halo; CN; CO₂H; CO₂ C₁₋₄alkyl;C(O)C₁₋₄alkyl; NH(R^(q)″); aryl(R^(a))₃; heteroaryl(R^(a))₃;NHC₁₋₄alkyl; N(C₁₋₄alkyl)₂; CONH₂; SH; S(O)_(y) C₁₋₆alkyl(R^(a))₃;C(O)NH C₁₋₆alkyl (R^(a))₃; C(O)N(C₁₋₆alkyl(R^(a))₃)₂; C₃₋₈cycloalkyl;NHC(NH)NH₂; heteroalkyl(R^(a))₃; —NHC(O)NH₂;

wherein

independently represent mono or bicyclic ring systems, non-aromatic orpartially aromatic, containing from 5-10 ring atoms, 1-4 of which are Nand 0-1 of which are O or S(O)_(y), with y equal to 0, 1 or 2,optionally containing 1-2 carbonyl groups; each R^(a) independentlyrepresents a member selected from the group consisting of: H, C₁₋₆alkyl, OC₁₋₆ alkyl, aralkyl, substituted aralkyl, heteroaralkyl,substituted heteroaralkyl, aralkoxy, substituted aralkoxy, halo,hydroxy, CN, CONH₂, CONHC₁₋₆ alkyl, CON(C₁₋₆ alkyl)₂, CO₂H, CO₂C₁₋₆alkyl, C(O)C₁₋₆alkyl, phenyl, CF₃, SH, NO₂, SO_(y) C₁₋₆alkyl, with y asdefined above; SO₂NH₂, SO₂NH C₁₋₆alkyl, NH SO₂(substituted aryl),NHSO₂(substituted heteroaryl), NH SO₂C₁₋₆ alkyl, NH SO₂aryl, NHSO₂heteroaryl, NH₂, NHC₁₋₆alkyl, N(C₁₋₆alkyl)₂, NHC(O)C₁₋₆alkyl,NHC(O)NH(C₁₋₆alkyl), C₂₋₄alkenyl and C₂₋₄ alkynyl; R^(q)′ represents H,OH, C₁₋₄alkyl, —O C₁₋₄alkyl, aryl or C(O) C₁₋₄alkyl, and R^(q)″represents H, OH or O C₁₋₄alkyl.
 2. A method according to claim 1wherein one or two R″ groups are present, and each independentlyrepresents NH₂, NHC₁₋₆alkyl(R^(q))₃, N(C₁₋₆alkyl)₂, NHC₃₋₈cycloalkyl,N(R^(q)′)C(O)C₁₋₆alkyl(R^(q))₃, C₁₋₆alkyl(R^(q))₃, OC₁₋₆alkyl(R^(q))₃,CO₂H, CONH₂, NR^(q)′C(O)NH C₁₋₆alkyl(R^(q))₃ or heterocyclyl(R^(q))₃. 3.A method according to claim 1 wherein: HETCy represents a 5-6 memberednon-aromatic heterocycle with 1-2 nitrogen atoms contained therein.
 4. Amethod according to claim 1 wherein HETCy represents a pyrrolidinyl orpiperidinyl group.
 5. A method according to claim 1 wherein R′ isselected C₁₋₆ alkyl(R^(q))₃, OC₁₋₆alkyl(R^(q))₃, C(O)CC₁₋₆alkyl(R^(q))₃; CN, NO₂ and CO₂ C₁₋₆alkyl(R^(q))₃.
 6. A method accordingto claim 1 wherein from 1-3 R groups are present and each independentlyrepresents a member selected from the group consisting of: halo,hydroxy, C₁₋₆alkyl(R^(q))₃, C₁₋₆alkyl(R^(q))₃, NH₂, NHC₁₋₆alkyl(R^(q))₃, N(C₁₋₆alkyl(R^(q))₃)₂ and CF₃.
 7. A method accordingto claim 6 wherein one or two R groups are present, selected from haloand CF₃.
 8. A method according to claim 1 wherein R^(x) is H,C₃₋₈cycloalkyl or C₁₋₆alkyl(R^(q))₃.
 9. A method according to claim 8wherein R^(x) represents H, CH₃, CH₂CH₃, CH₂CH₂CH₃, CH₂ CH₂CH₂ CH₃,

or


10. A method according to claim 1 wherein X′ represents a direct bond.11. A method according to claim 1 wherein X represents —(CH₂)_(m)—Y—(CH2)_(n)—, Y represents a direct bond, O, S or C(O); m represents 0or 1 and n represents 0 or
 1. 12. A method according to claim 11 whereinX represents a direct bond.
 13. The method according to claim 1 whereinthe CNS injury is ischemic stroke.
 14. The method according to claim 1wherein the CNS injury is caused by surgery, or is an open head injury.15. The method according to claim 1 wherein the CNS injury is a closedhead injury.